Isolated luciferases and the use thereof

ABSTRACT

The invention relates to the nucleotide and amino acid sequences, and to the activity and use, of the luciferases LuAL, Lu164, Lu16, Lu39, Lu45, Lu52 and Lu22.

The invention relates to the nucleotide and amino acid sequences, and tothe activity and use, of the luciferases LuAL, Lu164, Lu16, Lu39, Lu45,Lu52 and Lu22.

Luciferases

Luminescence is the term given to the emission of photons in the visiblespectral range, with this emission being brought about by excitatedemitter molecules. In contrast to fluorescence, the energy for this isnot supplied externally in the form of radiation of shorter wavelength.

A distinction is made between chemiluminescence and bioluminescence.Chemoluminescence is the term given to a chemical reaction which leadsto an excited molecule which itself emits light when the excitedelectrons return to the normal energy level. Bioluminescence is the termused when this reaction is catalyzed by an enzyme. The enzymes whichparticipate in the reaction are generally termed luciferases.

A review of luminescent organisms can be found in Hastings et al. 1995.

Luciferases are peroxidases or monooxygenases and dioxygenases. Theenzyme substrates, which form the starting substances for thelight-emitting products, are termed luciferins. They differ from speciesto species. The quantum yield of the systems lies between 0.1 and 0.9photons per transformed substrate molecule (Idelgaufts, 1993).

Luciferases can be classified on the basis of their origin or theirenzymic properties. An overview of some luciferase types is given below:

Bacterial Luciferase

TABLE 1 Bacterial luciferases Gene References/ product OrganismSubstrate □ Expression Patents Lux Vibrio FMN, 495 cytosolic Apley etal., Genes fischerii Dodecanal, nm 1985 NADH Gustafson G., U.S. Pat. No.5,196,524Coelenterazine-Dependent Eukaryotic Luciferases

TABLE 2 Coelenterazine-dependent eukaryotic luciferases Gene productOrganism Substrate □ Expression References/Patents Renilla RenillaCoelenterazine 480 nm cytosolic Mathews et al., Luciferase reniformis1977 Lorenz et al, 1991 Lorenz et al. 1996 Alan, P; WO 0020619 MiltonJ., U.S. Pat. No. 5,418,155 Roelant C., WO 9938999 Vargula/ VargulaVargula 460 nm secretory Thomspon et al., Cypridia hilgendorferiiLuciferin 1989 Luciferase Thompson et al., 1990 Tora, JP 05064583 Tora,JP 08027200 Renard et al., WO 9520653 Watasemia Watasenia Watasemia ?cytosolic Inoue et al., 1976 Luciferase scintillans Luciferin OlophorusOlophorus Coelenterazine 454 secretion Inouye et al, 2000 Luciferasegracilirostris Aequorin Aequoria Coelenterazine 470 nm cytosolic Head etal. 2000 aequoria (Ca²⁺ Shimomura et al., activated) 2000 Jones et al.,1999 Kendall et al., 1998 Inouye et al., 1985 Shimomura et al., 1969Cormier et al., U.S. Pat. No. 5,798,441 Cormier et al., U.S. Pat. No.5,422,266 Obelin Obelia Coelenterazine 470 nm cytosolic Matveev et al.,1999 Berestovskaya, 1999Coelenterazine-Independent Eukaryotic Luciferase

TABLE 3 Coelenterazine-independent eukaryotic luciferases Gene productOrganism Substrate □ Expression References Firefly Photinus Firefly 550cytosolic Webster et al., Luciferase pyralis Luciferin, nm 1980 ATPGould et al., 1988 Sala-Newby et al, 1992 Bonin et al., 1994 Sherf B.,U.S. Pat. No. 5,670,356 KIKK, JP 09187281

Luciferases can also be distinguished from each other on the basis oftheir substrate specificity. The most important substrates includecoelenterazine (Jones et al., 1999) and luciferin, and also derivativesof the two substances. Diagrams of the substrates, and theirtransformation by luciferase, are shown below:

Luciferase Substrates

Some luciferase substrates, and their transformation, are depicted belowby way of example. All the substrates which are shown here aretransformed enzymically with the release of light and carbon dioxide(CO₂) and consumption of oxygen (O₂). The dependence of the reaction oncofactors or energy carriers (e.g. ATP in the case of FireflyLuciferase) is enzyme-specific.

Coelenterazine

-   -   Transformation of coelenterazine into coelenteramide by aequorin        with the emission of light of wavelength 470 nm.

-   -   Transformation of luciferin into oxyluciferin by Firefly        Luciferase with the emission of light of wavelength 560 nm.

-   -   Transformation of Vargula Luciferin into Vargula Oxyluciferin by        Vargula Luciferase with the emission of light of wavelength 460        nm.        Reporter Systems

A reporter gene or indicator gene is the term which is generally givento genes whose gene products can readily be detected using simplebiochemical or histochemical methods. At least 2 types of reporter geneare distinguished.

-   -   1. Resistance genes. Resistance genes is the term given to genes        whose expression confers on a cell resistance to antibiotics or        other substances whose presence in the growth medium leads to        cell death when the resistance gene is absent.    -   2. Reporter gene. In recombinant DNA technology, the products of        reporter genes are used as fused or unfused indicators. The most        common reporter genes include beta-galactosidase (Alam et al.,        1990), alkaline phosphatase (Yang et al., 1997; Cullen et al.,        1992), luciferases and other photoproteins (Shinomura, 1985;        Phillips G N, 1997; Snowdowne et al., 1984).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows: Plasmid maps of the vectors pTriplEX2-LuAL, pcDNA3-LuALand pASM-LuAL.

FIG. 2 shows: Plasmid maps of the vectors pTriplEX2-Lu164, pcDNA3-Lu164and pASM-Lu164.

FIG. 3 shows: Plasmid maps of the vectors pTriplEX2-Lu22, pcDNA3-Lu22and pASM-Lu22.

FIG. 4 shows: Coelenterazine derivatives as potential substrates forLu164. A. graphic representation of the luminescence which was measured,at 8.7 kV for 30 seconds, in a luminometer (RLU, relative light units);B. diagrams of the molecular structures of the coelenterazinederivatives.

FIG. 5 shows: Emission spectra of luciferases Lu164 (A), LuAL (B) andLu22 (C) following bacterial expression. (RLU, relative light units).

FIG. 6 shows: Luciferase activity in CHO cell medium (5 82 l) followingtransfection of the cells with pcDNA3-LuAL, pcDNA3-Firefly, pcDNA3-Lu164and pcDNA3 as the control vector without any cDNA insertion. (RLU,relative light units; h, hours; Firefly: Firefly luciferase).

FIG. 7 shows: Temperature-dependent luciferase activity in CHO cellmedium (5 μl) following transfection of the cells with pcDNA3-LuAL,pcDNA3-Firefly and pcDNA3-Lu164. (RLU: relative light units; Medium:DMEM-F12+10% FCS).

FIG. 8 shows: Induced expression of LuAL in CHO cells. The cells wereinduced with Forskolin (10-5 M) for 5 hours at 37° C. The activity wasmeasured in 10 μl of cell supernatant (RLU: relative light units;induction factor: ratio of induced RLU to uninduced RLU).

FIG. 9 shows: Use of the luciferases as reporter genes for cellularsystems taking as an example the G protein-coupled receptors A2A andNPY2. (RLU: relative light units).

Classification of the Species Metridia longa

Arthropoda→→Crustacea→→Copepoda

The species Metridia longa belongs to the crustacea, especially thecopepoda or zooplancton.

Isolating the cDNA

In order to investigate the bioluminescence activity of the speciesMetridia longa, specimens were caught in the White Sea (KarteshBiological Station, Russia) and stored in liquid nitrogen. In order toprepare cDNA libraries of Metridia longa, the RNA was isolated by themethod of Krieg (Krieg et al., 1996) using isothiocyanate. RT-PCR wascarried out in order to prepare the cDNA. For this, 10 μg of RNA wereincubated with reverse transcriptase (Superscript Gold II) in accordancewith the following scheme:

PCR 1. 30 seconds 95° C. 2. 6 minutes 68° C. 3. 10 seconds 95° C. 4. 6minutes 68° C. 17 cycles of step 4 after step 3

In order to inactivate the polymerase, the reaction products wereincubated with proteinase K at 37° C. for 30 minutes, and the cDNA wasprecipitated with ethanol. The cDNA was dissolved in water and incubatedat 37° C. for one hour with SfiI. The reaction products were subjectedto gel filtration in order to separate off small fragments. Thefractionated cDNA was then ligated into the SfiI-cut anddephosphorylated □TriplEx2 vector. In order to prepare a □ phageexpression library, the cloned cDNA fragments were subsequently packagedinto □ phages using the SMART cDNA Library Construction Kit (Clontech)in-vitro packaging system.

The recombinant phages which contained a cDNA insertion with potentialfor expressing coelenterazine-dependent luciferases were identified bycarrying out a library screening.

For this, bacterial lawns composed of E. coli XL1-Blue were plated outon 90 mm culture dishes and incubated at 37° C. for 10 hours. They werethen infected with 2500 phages per culture dish, with this then beingfollowed by an incubation phase of 8 hours at 37° C. to enable plaquesto be formed. The culture dishes were subsequently stored at 4° C. forone hour in order to harden the soft agar.

In order to carry out a replica plating, nitrocellulose membranes weresaturated with E. coli XL1-Blue suspensions and dried. The dry membraneswere laid for 60 seconds on the phage plaques and then laid on freshagar plates. The agar plates were then incubated at 37° C. for 2 hoursand 4 ml of SOB medium (+10 mM MgSO4, 0.2% maltose) were added. Thebacterial lawn was detached, resuspended in LB medium (+20 mM IPTG) andincubated at 37° C. for one hour. The bacteria were harvested bycentrifugation and disrupted by ultrasonication, and the bioluminescenceactivity was determined in a luminometer after having addedcoelenterazine.

Culture plates giving a positive bioluminescence signal were dividedinto sectors and a fresh replica plating was carried out. The replicaplating was continued until active individual plaques had beenidentified. In order to subclone the cDNA insertions in the phages inpositive plaques [lacuna] took place into the pTriplEX2 vector in E.coli BM25.8 in accordance with the manufacturer's protocol for the SMARTcDNA library construction kit. The pTriplEx2 cDNA-transfected E. coliwere incubated overnight, at 37° C., in LB medium containing anampicillin concentration of 100 μg/ml. In order to achieveoverexpression, the overnight culture was diluted 1:150 with LB mediumand incubated at 37° C. for 1 hour. Induction was then effected byadding IPTG (isopropylthiogalactoside) to a final concentration of 20mM. The induced culture was incubated at 37° C. for 1 hour and thebacteria were harvested by centrifugation. The cells were disrupted byultrasonication in 0.5 ml of SM buffer. The chemiluminescence wasmeasured in a luminometer after adding 10 μl of coelenterazine (10⁻⁴ Min methanol).

Three luciferases which exhibited coelenterazine-dependent luciferaseactivity were identified. The luciferases were designated Lu164, LuALand Lu22. The luciferases are described in detail below.

The invention also relates to functional equivalents of the threeluciferases. Functional equivalents are those luciferases which have acomparable substrate spectrum, which are secreted and which are at least70% homologous. A homology of 80% or 90% is preferred. A homology of 95%is particularly preferred.

The luciferases are suitable for use as reporter genes for cellularsystems, especially for receptors, for ion channels, for transporters,for transcription factors or for inducible systems.

The luciferases can be used in bacterial systems, for example for titerdetermination or as substrates for biochemical systems, especially forproteinases.

The luciferases can also be used as reporter enzymes which are coupledto antibodies or other proteins, e.g. for ELISA, forimmunohistochemistry or for Western blotting.

The luciferases can be used in BRET (Bioluminescence Resonance EnergyTransfer) systems.

The luciferases are also suitable for use as fusion proteins forconfocal microscopy or for analyzing protein-protein interactions.

The luciferases can be used as reporter enzymes which are coupled tobiotin, NHS, CN—Br or other coupling mediators, e.g. for ELISA or forimmobilization.

The luciferases can furthermore be used as reporter enzymes which arecoupled to DNA or RNA oligonucleotides, e.g. for Northern and Southernblotting or for real time PCR.

The invention also relates to the purification of the luciferases aswild-type or tag proteins, and to the use of the luciferases in in-vitrotranslation systems.

Nucleotide and Amino Acid Sequences

LuAL

The luciferase LuAL is a protein having a molecular weight of 23.7 kDaand an isoelectric point of 8.32. The coding nucleotide sequence is:

5′atggatatgagggttatctttgctcttgttttctcatcattggttcaggccaaatcaactgaattcgatcctaacattaacattgttggtttagaaggaaaatttggtataacaaaccttgagacggatttattcacaatatgggagacaatggatgtcatcaaatcagatattacagatactgatagagtcagcaactttgttgcaactgaaaccgatgctaaccgtgggaaaatgcctggcaaaaaactgccactggcagttatcatggaaatggaagccaatgctttcaaagctggctgcaccaggggatgccttatctgtctttcaaaaataaagtgtacagccaaaatgaaggtgtacattccaggaagatgtcatgattatggtggtgacaagaaaactggacaggcaggaatagttggtgcaattgttgacattcccgaaatctctggatttaaggagatggcacccatggaacagttcattgctcaagttgatctttgcgctacctgcactactggatgtctcaaaggtcttgccaatgttaagtgctctgaactcctgaagaaatggctgcctggcagatgtgcaagttttgctgacaagattcaaaaagaagttcacaatatcaaaggcatggctgga gatcgttga 3′which gives rise to the following amino acid sequence:

MDMRVIFALVFSSLVQAKSTEFDPNINIVGLEGKFGITNLETDLFTIWETMDVIKSDITDTDRVSNFVATETDANRGKMPGKKLPLAVIMEMEANAFKAGCTRGCLICLSKIKCTAKMKVYIPGRCHDYGGDKKTGQAGIVGAIVDIPEISGFKEMAPMEQFIAQVDLCATCTTGCLKGLANVKCSELLKKWLPGRCASF ADKIQKEVHNIKGMAGDRand the following amino acid composition:

Ala: 18 (8.3%) Cys: 10 (4.6%) Asp: 14 (6.4%) Glu: 12 (5.5%) Phe: 10(4.6%) Gly: 19 (8.7%) His:  2 (0.9%) Ile: 18 (8.3%) Lys: 21 (9.6%) Leu:15 (6.9%) Met: 10 (4.6%) Asn:  8 (3.7%) Pro:  7 (3.2%) Gln:  5 (2.3%)Arg:  7 (3.2%) Ser:  9 (4.1%) Thr: 15 (6.9%) Val: 14 (6.4%) Trp:  2(0.9%) Tyr:  2 (0.9%)Lu 164

Luciferase Lu164 is a protein having a molecular weight of 23.8 kDa andan isoelectric point of 7.81. The coding nucleotide sequence is:

5′atggatataaaggttgtctttactcttgttttctcagcattggttcaggcaaaatcaactgaattcgatcctaacattgacattgttggtttagaaggaaaatttggtataacaaaccttgagacggatttattcacaatatgggagacaatggaggtcatgatcaaagcagatattgcagatactgatagagccagcaactttgttgcaactgaaaccgatgctaaccgtggaaaaatgcctggcaaaaaactgccactggcagttatcatggaaatggaagccaatgctttcaaagctggctgcaccaggggatgccttatctgtctttcaaaaataaagtgtacagccaaaatgaaggtgtacattccaggaagatgtcatgattatggtggtgacaagaaaactggacaggcaggaatagttggtgcaattgttgacattcccgaaatctctggatttaaggagatggcacccatggaacagttcattgctcaagttgaacgttgcgcttcctgcactactggatgtctcaaaggtcttgccaatgttaagtgctctgaactcctgaagaaatggctgcctgacagatgtgcaagttttgctgacaagattcaaaaagaagttcacaatatcaaaggcatggct ggagatcgttga 3′which gives rise to the following amino acid sequence:

MDIKVVFTLVFSALVQAKSTEFDPNIDIVGLEGKFGITNLETDLFTIWETMEVMIKADIADTDRASNFVATETDANRGKMPGKKLPLAVIMEMEANAFKAGCTRGCLICLSKIKCTAKMKVYIPGRCHDYGGDKKTGQAGIVGAIVDIPEISGFKEMAPMEQFIAQVDRCASCTTGCLKGLANVKCSELLKKWLPDRCAS FADKIQKEVHNIKGMAGDRand the following amino acid composition:

Ala: 21 (9.6%) Cys: 10 (4.6%) Asp: 15 (6.8%)  Glu: 13 (5.9%)  Phe: 10(4.6%) Gly: 18 (8.2%) His: 2 (0.9%) Ile: 18 (8.2%)  Lys: 22 (10.0%) Leu: 14 (6.4%) Met: 10 (4.6%)  Asn: 7 (3.2%) Pro:  7 (3.2%) Gln:  5(2.3%) Arg: 7 (3.2%) Ser: 8 (3.7%) Thr: 14 (6.4%) Val: 14 (6.4%) Trp: 2(0.9%) Tyr: 2 (0.9%)Lu22

Luciferase Lu22 is a protein having a molecular weight of 20.2 kDa andan isoelectric point of 7.89. The coding nucleotide sequence is:

5′atgggagtcaaacttatttttgctgttgtttgtgtcgcagttgcccaggctgccacaattcaggaaaattttgaagacattgatcttgtagccataggtggcagctttgcatcagatgttgatgctaacagaggtggacatggtggacatcctggcaaaaagatgccaaaagaagtacttatggaaatggaagccaatgctaaacgagctggctgccacaggggttgtctggtttgtctgtcacacatcaagtgcacagcacaaatgcagaagtttatcccaggaagatgccatagttatgcaggagacaaggattctgctcagggaggaattgccggtggtgccattgttgatatacctgaaattgccggatttaaagaaatgaagcccatggaacagttcattgctcaagttgatctctgtgaagattgcacaactggatgcctcaaaggtcttgccaatgttcattgctctgatctcctgaagaagtggctgccatcaagatgtaagacatttgcttccaaaattcaatctcaagtggataccatcaaaggtttggctggagatcgttga 3′which gives rise to the following amino acid sequence:

MGVKLIFAVVCVAVAQAATIQENFEDIDLVAIGGSFASDVDANRGGHGGHPGKKMPKEVLMEMEANAKRAGCHRGCLVCLSHIKCTAQMQKFIPGRCHSYAGDKDSAQGGIAGGAIVDIPEIAGFKEMKPMEQFIAQVDLCEDCTTGCLKGLANVHCSDLLKKWLPSRCKTFASKIQSQVDTIKGLAGDRand the following amino acid composition:

Ala: 21 (11.1%) Cys: 11 (5.8%) Asp: 12 (6.3%)  Glu: 9 (4.7%) Phe: 7(3.7%) Gly:  21 (11.1%) His: 6 (3.2%) Ile: 13 (6.8%)  Lys: 16 (8.4%) Leu: 12 (6.3%) Met: 7 (3.7%) Asn: 4 (2.1%) Pro: 6 (3.2%) Gln:  9 (4.7%)Arg: 6 (3.2%) Ser: 9 (4.7%) Thr: 6 (3.2%) Val: 13 (6.8%) Trp: 1 (0.5%)Tyr: 1 (0.5%)These sequences are also given in the sequence listing.Enzymic Activity and Biochemical Characterization of the Luciferases

The proteins LuAL, Lu164 and Lu22 are enzymes which release light whiletransforming coelenterazine. They therefore belong to the luciferases.The luciferases can be actively expressed in both bacterial andeukaryotic cells. The luciferases LuAl, Lu164 and Lu22 which areexpressed in eukaryotic cells are secreted. No secretion takes place inconnection with bacterial expression.

The activity of the luciferases is temperature-dependent. Temperatureoptima of 22° C. (for LuAL) and 27° C. (for Lu164) were determined forthe luciferases LuAL and Lu164, respectively. The temperature optimumfor luciferase Lu22 activity is 4° C. or lower.

EXAMPLES

Plasmids/Constructs

The vectors employed for preparing the constructs which are describedbelow were the vectors pcDNA3.1(+) and pTriplEx2 from Clontech and thevector pASMplr (in-house construct possessing cAMP-sensitive promoterelements; cre). The derivatives of the vectors were designated pcDNA3-x,pTriplEx2-x and pASM-x.

LuAL

FIG. 1 shows the plasmid maps of the vectors pTriplEX2-LuAL, pcDNA3-LuALand pASM-LuAL

FIG. 2 shows the plasmid maps of the vectors pTriplEX2-Lu164,pcDNA3-Lu164 and pASM-Lu164

FIG. 3 shows the plasmid maps of the vectors pTriplEX2-Lu22, pcDNA3-Lu22and pASM-Lu22

Coelenterazine Derivates as Substrates of Lu164

In order to identify substrates for Lu164, 10 μl solutions of differentcoelenterazine derivatives (10⁻⁴ M) were in each case incubated with 10μl of supernatant from CHO-pcDNA3-Lu164 cell lines and the luminescencewas measured. The coelenterazines were obtained from Molecular Probes(USA).

No differences as compared with luciferase Lu164 were seen in the caseof luciferases LuAL and Lu22. Unmodified coelenterazine (Fig. B,coelenterazine a) was identified as being the optimal substrate forLu164, LuAl and Lu22.

FIG. 4 shows coelenterazine derivatives as potential substrates forLu164 and a graph of the measurement of luminescence for 30 seconds at8.7 kV in a luminometer (RLU, relative light units); and also a diagramof the molecular structures of the coelenterazine derivatives.

Enzymic Activity of the Luciferases Lu164, LuAL and Lu22 in Dependenceon Coelenterazine

Bacterial Expression

The bacterial expression took place in the E. coli strain BL21(DE3) as aresult of transforming the bacteria with the expression plasmidspTriplEX2-Lu164, pTriplEX2-LuAL and pTriplEX2-Lu22. The transformedbacteria were incubated at 37° C. for 3 hours in LB medium andexpression was induced for 4 hours by adding IPTG to a finalconcentration of 1 mM. The induced bacteria were harvested bycentrifugation, resuspended in PBS and disrupted by ultrasonication.Coelenterazine (10⁻⁴ M in methanol) or luciferin (Firefly Luciferin) wasadded to 5 μl of the lysate (5 mg/ml) and the chemiluminescence wasmeasured.

The measurement, in RLU (relative light units), took place at 9.5 kV for30 seconds. Values of 230 000, 320 000 and 260 000 RLU were measured inthe case of Lu164, LuAL and Lu22, respectively. The enzymes wereexpressed in E. coli BL21(DE3) using the vectors pTriplEx2-Lu164,pTriplEx2-LuAL and pTriplEx2-Lu22.

Eukaryotic Expression

Constitutive eukaryotic expression was affected in CHO cells bytransfecting the cells with the expression plasmids pcDNA3-Lu164,pcDNA3-LuAL and pcDNA3-Lu22 in transient experiments. For this, 10 000cells in DMEM-F12 medium were plated, per well, in 96-well microtiterplates and incubated overnight at 37° C. The transfection was effectedusing the Fugene 6 kit (Roche) in accordance with the manufacturer'sinstructions. The transfected cells were incubated overnight at 37° C.in DMEM-F12 medium. The chemiluminescence in the medium (5 μl) and thecell lysate (5 μl) was measured for 30 seconds at 9.5 kV in aluminometer, at room temperature, after adding coelenterazine (10⁻⁴ M inmethanol).

Values of 680 000, 670 000 and 510 000 RLU (relative light units) weremeasured in the case of Lu164, LuAL and Lu22, respectively. Theexpression was effected in CHO cells using the vectors pcDNA3-Lu164,pcDNA3-LuAL and pcDNA3-Lu22.

Emission Spectra of the Luciferases Lu164, LuAL and Lu22

In order to measure the emission spectra, E. coli BL21(DE3) cells weretransformed with the plasmids pTriplEx2-Lu164, pTriplEx2-LuAL andpTriplEx2-Lu22 and overexpressed as described under 3.1. 50 μl ofcoelenterazine (10⁻⁴ M) were added to 100 μl volumes of the bacteriallysates and the emission spectra were measured. Graphs of the emissionspectra of the luciferases are shown below.

In the case of the luciferases LuAL, Lu164 and Lu22, maximum emissionresulting from the substrate transformation takes place at a wavelengthof about 490 nm.

FIG. 5 shows the emission spectra of the luciferases Lu164 (A), LuAL (B)and Lu22 (C) following bacterial expression (RLU, relative light units)

Secretion of the Luciferases Lu164, LuAL and Lu22 from CHO Cells, Takingas Examples Lu164 and LuAL

In order to characterize the expression of the luciferases LuAl, Lu164and Lu22 in eukaryotic cells, CHO cells were stably transfected with theplasmids pcDNA3-LuAl, pcDNA3-Lu164, pcDNA3-Fireluc and pcDNA3.1(+). Theresulting clones were cultured in DMEM-F12 medium. Firefly luciferasewas used as a positive control for nonsecreted luciferase. The plasmidpcDNA3.1(+) was used as a control plasmid for detecting potentialendogenous activity in the CHO parent cell.

In order to detect the secretion of the luciferases, 2000 cells wereplated on 384-well microtiter plates. After 24 hours, the medium wasremoved and the cells were washed with Tyrode solution and 30 μl offresh medium were added. The first measurement (0 h) then took place, ina luminometer at 9.5 kV for 30 seconds, after adding 5 μl ofcoelenterazine (10⁻⁴ M) or luciferin in the case of the Fireflyluciferase. The 1 h to 5 h measurements took place after one to fivehours.

FIG. 6 depicts the increase in luciferase activity in the medium independence on the time. The Firefly luciferase was not secreted. Theluciferases LuAL, Lu164 and Lu22 are secretory luciferases.

FIG. 6 shows the luciferase activity in the CHO cell medium (5 μl) afterthe CHO cells have been transfected with pcDNA3-LuAL, pcDNA3-Firefly,pcDNA3-Lu164 or pcDNA3 as the control vector without any cDNA insertion.(RLU, relative light units; h, hours; Firefly: Firefly luciferase)

Dependence of the Luciferase Activity on the Temperature

In order to determine the temperature dependence of the luciferasesLu22, Lu164 and LuAL, CHO cells were transiently transfected with thevectors pcDNA3-Lu22, pcDNA3-Lu164 and pcDNA3-LuAl and the luciferaseactivity in the supernatants was determined at temperatures of between 0and 47° C. In order to do this, the cell supernatant and thecoelenterazine solution were adapted to the measurement temperature for5 minutes. The measurement took place at 9.5 kV for 30 seconds in aluminometer.

FIG. 7 shows the luminescence which was measured, in dependence on thetemperature, in the case of the luciferases LuAl, Lu164 and Lu22. Thetemperature optimum for the luciferase activity of LuAL is 27° C. Atemperature optimum of 22° C. and of 4° C. or lower was determined inthe case of Lu164 and Lu22, respectively.

FIG. 7 shows the temperature-dependent luciferase activity in CHO cellmedium (5 μl) following transfection with pcDNA3-LuAL, pcDNA3-Fireflyand pcDNA3-Lu164. (RLU: relative light units; medium: DMEM-F12+10% FCS)

Induced Expression of the Luciferases Lu164, LuAL and Lu22 in CHO CellsTaking as an Example LuAL

Eukaryotic expression was induced in CHO cells by transfecting the cellswith the expression plasmid pASM-LuAL in transient experiments. Forthis, 10 000 cells in DMEM-F12 medium were plated per well in 96-wellmicrotiter plates and incubated overnight at 37° C. The transfection waseffected using the Fugene 6 kit (Roche) in accordance with themanufacturer's instructions. The transfected cells were incubatedovernight, at 37° C., in DMEM-F12 medium. They were then induced withForkolin (10⁻⁵ M) for 5 hours. The chemiluminescence in the medium andin the cell lysate was then measured, at 9.5 kV for 30 seconds, in aluminometer after having added coelenterazine (10⁻⁴ M in methanol).

FIG. 8 shows the induced expression of LuAL in CHO cells. The expressionwas induced for 5 hours with Forskolin (10⁻⁵ M) at 37° C. The activitywas measured in 10 μl of cell supernatant (RLU: relative light units;induction factor: ratio of induced RLU to uninduced RLU)

Use of the Luciferases Lu164, LuAL and Lu22 as Reporter Genes inCellular Systems Taking as Examples the Receptors NPY2 and A2A and UsingLuAL as the Reporter Gene

In order to be able to analyze the activation of G protein-coupledreceptors by receptor-specific ligand in cell-based systems, the cDNAsequence for luciferase LuAL was cloned into the expression vectorpASMplr. The expression vector pASMplr contains cAMP-sensitive promoterelements (CRE) which enable the intracellular concentration of cAMP tobe measured indirectly. The luciferase serves as the reporter gene inthe system.

The use of the luciferases Lu22, Lu164 and Lu22 as reporter genes incellular systems was demonstrated by taking as an example the Gprotein-coupled receptors NPY2 (neuropeptide receptor 2) and A2A(adenosine receptor 2a). To do this, the stable clone CHO-pASM-LuAL wastransiently transfected with the vector pcDNA3-NPY2 or the vectorpcDNA3-A2A. The receptor NPY2 is a Gi-coupled receptor, while the A2Areceptor is a Gs-coupled receptor.

The A2A receptor was activated for 4 h by adding 1 μM NECA. The NPY2receptor was activated by adding 10 μM NPY2 peptide in the presence of10⁻⁵ M Forskolin. The luciferase activity in the medium (30 μl) wasmeasured, at 9.5 kV and for 30 seconds in a luminometer, after havingadded coelenterazine (10⁻⁴ M).

FIG. 9 shows the use of the luciferases as reporter genes for cellularsystems taking as an example the G protein-coupled receptors A2A andNPY2. (RLU: relative light units)

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What is claimed is:
 1. An expression vector comprising an isolatednucleic acid molecule selected from the group consisting of: (a) anucleic acid molecule comprising SEQ ID NO: 1; and (b) a nucleic acidmolecule which exhibits at least 90% sequence identity to the nucleicacid molecule of SEQ ID NO: 1, and wherein said nucleic acid moleculeencodes luciferase.
 2. A transformed host cell comprising the expressionvector of claim 1.